3 Before starting Prepare all components and get all necessary materials ready by examining this instruction booklet and become familiar with each steps. Important Determine the volume of Buffer LY to be used and add 20 µl of β-mercaptoethanol (β-me) per 1 ml Buffer LY before use. Bufer LY contains β -ME can be stored at room temperature for up to 1 month. Crystals may form in Buffer LY, dissolve the precipitates at 37 o C before use. Add 48 ml (R01-01, R01-05) or 200 ml (R01-02, R01-06) 100% ethanol to RNA Wash Buffer before use. The final ethanol content is 80% (v/v). DNase I preparation For each column, prepare the DNase I digestion reaction mixture as follows: Materials supplied by users DNase I Digestion Buffer 73.5 µl RNase-free DNase I (20 Kunits units/ µl) 1.5 µl Total volume 75 µl Tabletop microcentrifuge and 1.5 ml sterile tubes. Vacuum manifold if use vacuum protocol. 100% ethanol. β-mercaptoethanol. Disruption and homogenization of tissue samples It is critical to disrupt and homogenize the samples completely and properly for high quality RNA yield. The purpose for homogenization is to reduce the viscosity by shearing genomic DNA and other high molecular weight cell components to create a homogenous lysate. Incomplete homogenization may result in clogging the column and reducing the RNA yield. 1. Sample disruption by mortar and pestle Excise tissues and freeze in liquid nitrogen immediate. Grind the sample with ceramic mortar and pestle to a fine powder under liquid nitrogen. Transfer the suspension into a tube pre-chilled in liquid nitrogen and allow the liquid nitrogen to evaporate while the samples remain frozen. Add Buffer LY before the sample gets thawed. 2. Homogenization using homogenization columns It is a fast and efficient way to homogenize the samples using Bioland s RNA Shredders. Up to 700 μl of samples can be loaded per column. Homogenization columns are supplied in the Plant RNA Kit and can be purchased separately for use with the tissue RNA kit. 3. Rotor-Stator for sample disruption and homogenization Using a proper size probes and generator, the process simultaneously disrupts and homogenizes most of samples. 4. Bead milling for sample disruption and homogenization Cells and tissues can be disrupted and homogenized by rapid agitation in the presence of glass beads in Buffer LY. Use 4-8 mm glass beads for animal tissues, 0.5 mm for yeast cells and 0.1 mm for bacterial samples. EasyPrep TM Total RNA Extraction 5 6 EasyPrep TM Total RNA Extraction

4 Removal of genomic DNA using DNase digestion DNA digestion is necessary for downstream applications that are sensitive to very small amounts of DNA, for example, RT-PCR with low-abundance target. Generally, it is not required to do so since the EasyPrep TM RNA purification kit selectively isolates RNA and eliminates most of the DNA. If there is DNA contamination, either reduces the tissue amount or cells. Stabilization of RNA in harvested animal tissues The intact of RNA in harvested tissue will be protected with the addition of RNA Secure solution (Catalog# R1011). 1. Cut the tissue into slices less than 0.5cm thick and immediately add at least 15 volumes of RNAsecure solution, for example, 150 μl RNAsecure solution per 10 mg tissue. 2. Store at room temperature for up to 24 hours, at 4 C for up to a week, and -20 C or -80 C for long term. RNA quality It is highly recommended that RNA quality be determined before downstream applications. The quality of RNA can be assessed by denatured agarose gel electrophoresis with the ethidium bromide staining. Several sharp bands should appear on the gel including 28S and 18S ribosomal RNA bands as well as certain populations of mrna and bands. If these bands smear towards lower molecular weight RNAs, then the RNA has undergone major degradation during preparation, handling or storage, RNA molecule less than 200 bases in length do not efficiently bind to the RNA column. An A260/ A280 ratio of corresponds to % pure nucleic acid. Table 1. Typical yield of total RNA per column Sample 10 mg/500 μl Buffer LY Total RNA Yield (μg) Liver 10 mg 50 (10 mg tissue) Kidney 10 mg (10 mg tissue) Muscle* 10 mg 20 (10 mg tissue) Spleen 10 mg (10 mg tissue) Heart* 10 mg 50 (10 mg tissue) Brain** 10 mg 80 (10 mg tissue) Lung 10 mg (10 mg tissue) Pancreas 10 mg 20 (10 mg tissue) HeLa Cells 1x (1x10 6 cells) 293HEK 1x (1x10 6 cells) COS-7 1 x (1x10 6 cells) NIH/3T3 1x (1x10 6 cells) *Note: It is normally difficult to isolate RNA from heart, muscle, and skin tissue using the regular RNA isolation procedure due to the rich contents of connective tissue, collagen, and contractile proteins. Optimization with the addition of proteinase K digestion that enables the removal of proteins described above is needed. **Note: For isolating RNA from lipid rich animal tissue, such as thymus and brain tissue, the yield will be low. Determine amounts of samples to be processed The yield depends on the tissue and cells to be processed. Please reference Table 1 to determine the amount of sample. EasyPrep TM Total RNA Extraction 7 8 EasyPrep TM Total RNA Extraction

5 Extracting total RNA from cultured cells 1. Cell preparations: (Do not use more than 5x10 6 of cells) Suspension cultured cells: Collect cells by centrifuging at 300 x g for 5 min. Remove all supernatant completely by aspiration. Adherent cultured cells: Aspirate the medium completely with a Pasteur pipet. Go to step 2 immediately by adding Buffer LY. Residual supernatant will inhibit cell lysis and affect the RNA yield. 2. Suspension cells: Flicking the tube to loosen the cell pellet and add 500 µl Buffer LY. Adherent cells: Add 500 µl Buffer LY directly into the dish. Use pipet tip to mix and transfer the cell lysate to a 1.5 ml tube. Add 20 µl of β-mercaptoethanol (β -ME) per 1 ml Buffer LY before use. Buffer LY contains β -ME can be stored at room temperature for up to 1 month. 3. Homogenize the lysate by vortexing vigorously or repeated pipetting until the sample is uniformly homogenized. If the solution is clear, go to step 5, otherwise go to step Centrifuge the solution at 13,000 rpm for 2 min and transfer the clear lysate to a clean 1.5 ml tube. 5. Add 1/2 volume 100% ethanol into the lysate (For example: 250 µl 100% ethanol for 500 µl lysate) and pipet 5 times to mix the solution. Vortex briefly if any precipitations. 6. Transfer the solution to a RNA column and centrifuge at 10,000 rpm for 1 min. Discard the flow-through. Without DNase I treatment: 7. Add 400 µl Buffer RB to the column and centrifuge at 10,000 rpm for 30s. Discard the collection tube with the flowthrough. Put the column back to a new collection tube. 10,000 rpm for 30 s. Discard the flow-through. Ensure that ethanol is added to RNA Wash Buffer before use. 9. Centrifuge the column with the lid open at 10,000 rpm for 1 min. It is critical to remove residue ethanol for optimal elution. 10. Transfer the column to an RNase-free 1.5 ml tube. Add µl DEPC-treated ddh 2 O to the center of the column. Centrifuge at 10,000 rpm for 1 min to elute the RNA. Store the RNA solution at -20 C. With DNase I treatment: 7. Add 500 µl RNA Wash Buffer to the column and centrifuge at 10,000 rpm for 30 s. Discard the flow-through. Ensure that ethanol has been added to RNA wash buffer before use. 8. Add 75 µl DNase I (5U,RNase-free) with Digestion Buffer onto the middle of the column and incubate at room temperature for 15 min. Add 400 µl Buffer RB to the column, incubate for 1-2 min, and centrifuge at 10,000 rpm for 30s. Discard the collection tube with the flow-through. Put the column back to a new collection tube. 9. Add 600 µl RNA Wash Buffer to the column and centrifuge at 10,000 rpm for 30 s. Discard the flow-through. Ensure that ethanol has been added to RNA wash buffer before use. 10. Centrifuge the column with the lid open at 10,000 rpm for 1 min. It is critical to remove residue ethanol for optimal elution. 11. Transfer the column to an RNase-free 1.5 ml tube. Add µl DEPC-treated ddh 2 O to the center of the column. Centrifuge at 10,000 rpm for 1 min to elute the RNA. Store the RNA solution at -20 C. 8. Add 600 µl RNA Wash Buffer to the column and centrifuge at EasyPrep TM Total RNA Extraction 9 10 EasyPrep TM Total RNA Extraction

6 Extracting Total RNA from Animal Tissue 1. Quickly weight an appropriate mass tissue according to Table 1 (Page 6) and transfer the tissue into a 1.5 ml tube containing 500 µl Buffer LY (add β-mercaptoethanol before use) and homogenize the tissue by a rotor starter or ultrasonic homogenizer on ice. Determine the volume of Buffer LY to be used and add 20 µl of β-mercaptoethanol (β-me) per 1 ml Buffer LY before use. Buffer LY contains (β-me) can be stored at room temperature for up to 1 month. Use of too much mass of tissue per preparation will cause genomic DNA contamination. 2. Centrifuge the lysate for 5 min at 13,000 rpm at room temperature and transfer the cleared lysate to a clean 1.5 ml tube. 3. Add 1/2 volume of the 100% ethanol to the lysate (for example: 250 μl 100% ethanol for 500 μl lysate). 4. Transfer the solution into a RNA column and centrifuge at 10,000 rpm for 1 min. Discard the flow-through liquid. Without DNase I treatment: With DNase I treatment: 5. Add 500 µl RNA Wash Buffer to the column and centrifuge at 10,000 rpm for 30 s. Discard the flow-through. Ensure that ethanol has been added to RNA wash buffer before use. 6. Add 75 µl DNase I (5U, RNase-free) with Digestion Buffer onto the middle of the column and incubate at room temperature for 15 min. Add 400 µl Buffer RB to the column, incubate for 1-2 min, and centrifuge at 10,000 rpm for 30s. Discard the collection tube with the flow-through. Put the column back to a new collection tube. 7. Add 600 µl RNA Wash Buffer to the column and centrifuge at 10,000 rpm for 30 s. Discard the flow-through. Ensure that ethanol has been added to RNA wash buffer before use. 8. Centrifuge the column with the lid open at 10,000 rpm for 1 min. It is critical to remove residue ethanol for optimal elution. 9. Transfer the column to an RNase-free 1.5 ml tube. Add µl DEPC-treated ddh 2 O to the center of the column. Centrifuge at 10,000 rpm for 1 min to elute the RNA. Store the RNA solution at -20 C. 5. Add 400 µl Buffer RB to the column and centrifuge at 10,000 rpm for 30s. Discard the collection tube with the flowthrough. Put the column back to a new collection tube. 6. Add 600 µl RNA Wash Buffer to the column and centrifuge at 10,000 rpm for 30 s. Discard the flow-through. Ensure that ethanol is added to RNA Wash Buffer before use. 7. Centrifuge the column with the lid open at 10,000 rpm for 1 min. It is critical to remove residue ethanol for optimal elution. 8. Transfer the column to an RNase-free 1.5 ml tube. Add µl DEPC-treated ddh 2 O to the center of the column. Centrifuge at 10,000 rpm for 1 min to elute the RNA. Store the RNA solution at -20 C. EasyPrep TM Total RNA Extraction EasyPrep TM Total RNA Extraction

7 RNA cleaning Protocol 1. Add 500 µl Buffer LY (add -mercaptoethanol before use) to the reaction (up to 100 µl). 2. Add 1/2 volume 100% ethanol into the mixture (for example: 250 µl 100% ethanol for 500 µl mixture) and pipet 5 times to mix the solution. Vortex briefly if any precipitations. 3. Transfer the solution into the binding column and centrifuge at 10,000 rpm for 1 min. Discard the collection tube with the flow-through and put the column back to a new collection tube. 4. Add 500 µl RNA Wash Buffer to the column and centrifuge at 10,000 rpm for 30 s. Discard the flow-through. 5. Optional: Add 75 µl DNase I (5U,RNase-free) solution onto the middle of the column and incubate at room temperature for 15 min. Add 400 µl Buffer RB onto the column and centrifuge at 10,000 rpm for 1 min. Discard the flow-through. Add 300 µl RNA Wash Buffer to the column and centrifuge at 10,000 rpm for 1 min. Discard the flow-through. 6. Add 500 µl RNA Wash Buffer to the column and centrifuge at 10,000 rpm for 30 s. Discard the flow-through. 7. Centrifuge the column with the lid open at 10,000 rpm for 1 min. Discard the flow-through. It is critical to remove residual ethanol for optimal elution. 8. Place the column to an RNase-free 1.5 ml tube. Add µl DEPC-treated water to the column, incubate for 1 min, and centrifuge at 10,000 rpm for 2 min. Store the RNA solution at C. Troubleshooting Problem Low A260/A280 ratios Low Yield Genomic DNA contamination Genomic DNA contamination Possible reason Protein contamination Guanidine Thiocyanate contamination RNA in sample degraded The binding capacity of the membrane in the spin column was exceeded Ethanol not added to buffer Too much total RNA sample was used in RT-PCR. The sample may contain too much genomic DNA. Suggested Improvement Do a Phenol:Chloroform extraction. Loss of total RNA (up to 40%) should be expected. Add 2.5 volumes of ethanol and 0.1M NaCl (final concentration) to precipitate RNA. Incubate for 30 min at 20 C. Centrifuge at 10,000 g for 15 min at 4 C. Resuspend the RNA pellet in DEPC-treated water. Freeze samples immediately in liquid nitrogen and store at -70 C after collect it. Use of too much tissue sample exceeding the binding capacity of spin column will cause the decreasing of total RNA yield. Add ethanol to the RNA Wash Buffer and DNase Stop Solution before purification. Reduce total RNA amount used in RT-PCR to ng. Reduce the amount of starting tissue in the preparation of the homogenate. Most tissues will not show a genomic DNA contamination problem at 30 mg or less per prep. Reduce cell numbers to 1-2x10 5 or increase buffer volume and do multiple loadings to column. EasyPrep TM Total RNA Extraction EasyPrep TM Total RNA Extraction

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